Project Summary. Osteoarthritis (OA) is a leading cause of disability in the industrialized world but there are no approved disease modifying drugs for OA. The long-term objective of my laboratory is to understand the factors that mediate the development and maintenance of articular cartilage so that specific targets to prevent or treat OA can be identified. TGF- is a multifunctional peptide that has been shown to regulate skeletal development and tissue-specific gene expression. We were the first to identify TGF- as a chondroprotective factor. The importance of TGF- in human osteoarthritis has now been well established. We previously identified several down-stream targets of TGF- that regulate post-translational processing of the major extracellular matrix proteins in cartilage. One in particular, 3-Prime-Phoshoadenosine 5-Prime-Phosphosulfate Synthase 2 (Papss2), has been associated with osteoarthritis in humans and mice and is required for proper sulfation of glycosamino glycans in cartilage. The transcription factor Sox9 has similar roles in cartilage as TGF- and in the last funding period we showed that TGF- regulates post-translational modifications on Sox9 and increases the half-life of the Sox9 protein. Furthermore, we showed that Sox9 is sufficient and required for TGF--mediated regulation of Papss2 transcription defining a novel signaling pathway for TGF- in cartilage. We also showed, using a bioreactor model, that expression of Sox9 in cartilage deficient for TGF- signaling was sufficient to restore proteoglycan staining to that cartilage. In this funding period, we propose to continue to determine the mechanisms by which TGF- post-translationally modifies the Sox9 protein and determine the biological consequences of the modifications. We will also determine the molecular mechanism whereby TGF- regulates Papss2 expression through a novel Sox9-dependent mechanism. Detailed molecular information about the mechanisms of TGF- action in articular cartilage will allow us to develop high throughput assays for future drug discovery screens. We propose the following specific aims: 1A) To determine how TGF- regulates the phosphorylation of Sox9 on Serine 181; 1B) Determine the role of Serine 211 in TGF--mediated stabilization of Sox9 protein; 1C) Determine the mechanism whereby TGF- treatment results in sumoylation of Sox9; 2) Determine the biological functions of post translationally modified Sox9 3) Identify TGF- /Sox9 responsive DNA elements in the Papss2 gene; and 4) Determine if downstream targets of TGF- can prevent cartilage degeneration and OA. The information acquired during this project period will provide cartilage unique targets for drug discovery to prevent or treat osteoarthritis.